Abstract : The milk fat globule membrane MFGM, the material surrounding milk fat globules, is not only interesting from a technological standpoint but it also shows great potential as a health ingredient, as it exerts cytotoxic and apoptotic effects against colon cancer cells. Although the effects of milk processing on the MFGM composition and functionality are well documented, less is understood on how processing may affect its bioactivity. This study aimed to determine if heating can affect the antiproliferative capacity of the MFGM. To do so, MFGM was extracted from milk heated at 80 °C for 10 min, as this temperature-time regime is known to cause extensive protein-protein interactions with changes in the processing functionality of milk. Two cell lines, whose morphological features are representative of two different stages of colon carcinogenesis HT-29 and Caco-2, were used to test the antiproliferative capacity of MFGM isolates obtained either from untreated or heated milk. Cell proliferation analysis showed a similar dose-dependent decrease of DNA synthesis in both cell lines exposed to 6.25–200 μg of MFGM protein.mL−1, isolated from unheated milk. The heat treatment diminished the efficacy of the MFGM isolates, as only the highest concentrations of MFGM tested following heating showed a significant effect on cell proliferation. The decreased ability of MFGM isolates to affect the carcinoma cell proliferation was attributed to changes in composition, mainly phospholipid losses. Changes to the supramolecular structure of the MFGM caused by heating may also have played a role. This work demonstrates the importance of processing history in assessing the biological functionality of MFGM.